Bottom Line:
Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin.This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle.We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.

Affiliation: Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Melbourne, Australia.

ABSTRACTPlasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.

ppat-1001292-g006: Hypothetical model for PfH2A.Z dynamics at the var transcription start site.(A) In rings, H3K4me3, H3K9ac and PfH2A.Z are present at the TSS of the var gene that is actively transcribed by the RNA PolII complex. (B) In schizonts, the active var gene is in a silent state poised for transcription in the next cycle. H3K4me3 at the promoter is demethylated to H3K4me2 [34]. H3K9 is deacetylated and PfH2A.Z removed, which might be mediated by the histone deacetylase Sir2A. (C) In parasites in which Sir2A is disrupted, H3K9ac and PfH2A.Z are maintained at the poised var promoters in schizont stages.

Mentions:
Possibly PfSir2A is not only involved in maintaining var gene silencing in heterochromatin by removing activating histone acetylations such as H3K9ac [35], but also assists in the temporary expulsion of PfH2A.Z from the active var promoter in mature parasites (Model in Figure 6). This could occur indirectly through recruitment of the ATP-dependent chromatin remodeling machinery responsible for histone variant exchange, or directly through Sir2-mediated deacetylation of PfH2A.Z. Sir2 plays such a direct role in regulating H2A.Z levels in human myocytes where over-expressed Sir2 deacetylates H2A.Z which in turn leads to ubiquitination and proteasome-dependent degradation of H2A.Z [106].

ppat-1001292-g006: Hypothetical model for PfH2A.Z dynamics at the var transcription start site.(A) In rings, H3K4me3, H3K9ac and PfH2A.Z are present at the TSS of the var gene that is actively transcribed by the RNA PolII complex. (B) In schizonts, the active var gene is in a silent state poised for transcription in the next cycle. H3K4me3 at the promoter is demethylated to H3K4me2 [34]. H3K9 is deacetylated and PfH2A.Z removed, which might be mediated by the histone deacetylase Sir2A. (C) In parasites in which Sir2A is disrupted, H3K9ac and PfH2A.Z are maintained at the poised var promoters in schizont stages.

Mentions:
Possibly PfSir2A is not only involved in maintaining var gene silencing in heterochromatin by removing activating histone acetylations such as H3K9ac [35], but also assists in the temporary expulsion of PfH2A.Z from the active var promoter in mature parasites (Model in Figure 6). This could occur indirectly through recruitment of the ATP-dependent chromatin remodeling machinery responsible for histone variant exchange, or directly through Sir2-mediated deacetylation of PfH2A.Z. Sir2 plays such a direct role in regulating H2A.Z levels in human myocytes where over-expressed Sir2 deacetylates H2A.Z which in turn leads to ubiquitination and proteasome-dependent degradation of H2A.Z [106].

Bottom Line:
Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin.This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle.We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.

Affiliation:
Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Melbourne, Australia.

ABSTRACTPlasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes.